After detecting infection or injury, the phagocytic cells of the immune system carry out an inflammatory response, which destroys invading microorganisms, clears debris from dead cells, and restores homeostasis. Neutrophils, in particular, have a critical role at every stage of the inflammatory response-they are the first cels to be recruited to the site of infection or injury; they kill microbes, engulf debris, and orchestrte the activities of other immune cells; and their apoptosis triggers resolution of inflammation and the restoration of tissue homeostasis. Inflammation is essential to fight infection and initiate healing, but prolonged or hyperactive inflammation may cause harm to healthy cells. Furthermore, chronic inflammation exacerbates diseases that place a huge burden on society, such as diabetes, atherosclerosis, and cancer. Our incomplete understanding of the mechanisms that control the initiation and resolution of inflammation has hindered our progress toward therapies for these debilitating conditions. The long-term goal of this project is to discover new genes with essential functions in regulating inflammation by analyzing zebrafish mutants with abnormalities in neutrophils. An increase in neutrophil number and recruitment of neutrophils to peripheral tissues are conserved hallmarks of inflammation, and an active neutrophil response is a robust indicator of inflammation. We propose to exploit the power of zebrafish genetics to understand the control of inflammation and neutrophil activity. Through the analysis of a zebrafish mutant with a hyperinflammatory phenotype, we have discovered a new gene that is essential to prevent inflammation in the absence of infection or injury. With the following three Specific Aims, we propose to isolate more mutations that disrupt inflammation and neutrophil activity, and to study the functions of these genes at the cellular and molecular level. These studies will define important new regulators of the inflammatory response and may thereby provide new avenues toward therapeutic control of inflammation. Aim 1: We will conduct a genetic screen with a neutrophil-specific transgenic reporter for mutations in new genes with essential functions in development, migration, and activity of neutrophils. Aim 2: To determine the functions of the mutated genes at the cellular level, we will characterize the mutant phenotypes by analyzing inflammatory signaling, determining which aspects of neutrophil activity and development are disrupted, and testing the neutrophil response to tissue injury. Aim 3: To discover the biochemical functions of the mutated genes, we will clone the mutated genes and analyze the distribution and activities of their products.